This project of the Hematopoiesis Section is focused on the basic biology of hematopoietic stem cells (HSC). HSC are a rare population of self-renewing cells that give rise to all cells in the peripheral blood. For patients with a life-threatening hematologic disease, transplantation of HSC from a healthy closely matched donor after ablation of the diseased bone marrow can be a life long cure. However, the main risk in these procedures is the transplantation of inadequate numbers of HSC. Our goal is to understand the processes that promote HSC self renewal and inhibit HSC differentiation. By manipulating the balance between self-renewal and differentiation we will be able to increase the number of stem cells and consequently increase the effectiveness of bone marrow transplantation to cure acquired or inherited hematopoietic diseases. [unreadable] Specific Aim 1: We have shown that activation of the canonical Wnt signalling pathway by Wnt3a is associated with increased HSC proliferation and eventually leads to increased differentiation of HSC. In contrast activation of a non-canonical Wnt signalling pathway by Wnt5a inhibits the canonical Wnt signalling pathway in HSC, resulting in HSC exiting the cell cycle and returning to a quiescent state which makes them better at repopulation. To dtermine the cells of the hematopoietic microenvironment responsible for production of these Wnt molecules, we are desigining transgenic mice in which either Wnt3a or Wnt 5a can be inducibly deleted in either hematopoietic or bone marrow stromal cells. We will use these Wnt KO animals as both donor and recipients to determine which cells and which Wnt molecules are responsible for engrafting and expanding transplanted HSC in vivo. We hypothesize that sequential treatment of HSC with first Wnt3a and then Wnt5a will expand HSC numbers before rendering them quiescent for transplantation. We will expose enriched HSC to Wnt3a to induce maximum cell cycling and then change to Wnt5a to return the cells to quiencence. By transplanting the cells during this time course we will determine the optimum timing of Wnt addition to induce the maximum increase in HSC. For enriched populations of human HSC we will perform similar experiments and follow the cell cycle status to determine whether the conditions identified for mouse can be applied to human HSC.[unreadable] Specific Aim 2: The bone marrow stromal cells are a mixed population of macrophages, endothelial and fibroblast cells that provides a matrix in which HSC become embeded. However, the maintainence of these cells in culture is not uniform and the different cytokines produced have differing effects on HSC. Near the trabecular bone where the density of the extracellular matrix is most dense, HSC are most actively undergoing self renewal. In the center of the marrow space, where the matrix is considerable softer, HSC are undergoing differentiation. To achieve a uniform enironment in which specific soluable factors can be added or subtracted we will evaluate stem cell survival and expansior or differentiation of collagen matrices of different densities designed to mimic the bone interface or the center of the marrow. We will compare the number of HSC growing on each matrix by competitive bone marrow transplantation and the ability to differentiate using in vitro colony forming assays. The goal ofthese studies is to devise an ex vivo culture condition to expand compatible donor HSC to numbers that can be safely transplanted. For enriched populations of human HSC we will perform similar experiments and follow the cell cycle status and colony forming abilities to determine whether the conditions identified for mouse can be applied to human HSC.